Molecular markers for chloroquine-resistant Plasmodium falciparum malaria in Thailand and Laos

Abstract

Chloroquine-resistant Plasmodium falciparum is well documented in Thailand. Laos, however, continues to use chloroquine (CQ) as the first-line therapy for the treatment of P. falciparum malaria. The objective of the present study was to determine the prevalence, in these two areas, of the cg2, pfmdr1 and pfcrt allelic types that have previously been associated with CQ resistance. Isolates of P. falciparum were collected from participants in ongoing treatment studies conducted in Thailand (near the Thai-Cambodian border) and in Laos (Vang Vieng district). The pfmdr1 and pfcrt alleles were characterized by PCR-RFLP and mutations in cg2 were characterized by PCR and single-stranded-conformation-polymorphism (SSCP) electrophoresis. Eight (32%) of the 25 Laotian isolates but only one (4%) of the 25 Thai isolates were found to contain the pfmdr1 mutation N86Y (P = 0.02). In contrast, the cg2 polymorphisms previously associated with CQ resistance were present in only 10 of the isolates from Laos but 24 of those from Thailand (40% v. 96%; P < 0.001). All the samples from both countries contained the pfcrt K76T mutant allele reported to confer resistance to CQ. The results may indicate that drug pressure for the maintenance of the pfmdr1 and cg2 alleles varies in intensity in the Thai and Laotian study areas, probably reflecting differences in the national malaria-treatment policies of Thailand and Laos.

Association of Plasmodium Falciparum Chloroquine Resistance Transporter Variant T76 With Age-Related Plasma Chloroquine Levels

“…This observation supports the view that the pfcrt polymorphism at position 76 is in fact a significant factor of CQ resistance, as shown in previous studies from Cameroon, 13 Mali, 8 Mozambique, 10 Nigeria, 20 Sudan, 12 Uganda, 9,21 Madagascar, 22 Laos, 11,23 Papua New Guinea, 14,24 and Thailand. 14,23 The proportion of children infected with parasite clones carrying the pfcrt T76 variant decreased significantly with age and correlated with the age-associated decrease in CQ levels. The higher levels of CQ in younger children might result from the fact that these children are more frequently given CQ when they are febrile during an assumed malaria attack.…”

Molecular characterisation of drug-resistant Plasmodium falciparum from Thailand

“…Our in vitro observations show a near total prevalence of CQR (96%) in the present study area, confirming what has been widely reported [1]. Among all CQR isolates tested in our study, the presence of pfcrt 76T was universal ( P = 0.001), indicating complete selection of this polymorphism by the drug, a result that is in agreement with recently published work not only from Thai-originated parasites [22], but also from parasites of different areas of the globe [32]. …”

pfmdr1 mutations associated with chloroquine resistance incur a fitness cost in Plasmodium falciparum

“…Several studies have shown that more than 50% of Thai isolates are resistant to MQ in vitro, and that the prevalence of the D10-like N1042 and D1246 pfmdr1 polymorphisms is greater than 90% (Price et al, 1999;Lopes et al, 2002;Price et al, 2004), consistent with both a fitness advantage in the absence of CQ and previous observations associating MQ resistance with these polymorphisms (Duraisingh et al, 2000b;Reed et al, 2000). Despite the prevalence of the wild-type (CQS) pfmdr1 allele, in vivo CQ resistance in Thailand remains high, probably as a consequence of the CQR-conferring K76T mutation in PfCRT having reached near-fixation throughout the country (Price et al, 1999;Labbe et al, 2001;Lopes et al, 2002).…”

“…Therefore in areas where the treatment for malaria includes MQ but not CQ, parasites with mutations at pfmdr1 codons 1034, 1042 and 1246 would be doubly disadvantaged with respect to parasites with the wild-type pfmdr1 allele, in terms of both decreased fitness and heightened drug sensitivity. This is borne out by recent findings in Thailand, where use of CQ as the first-line treatment for falciparum malaria was discontinued in 1972 in favour of Fansidar (a combination of the antifolate drugs sulfadoxine and pyrimethamine), which in turn was replaced by Fansimef (MQ with sulfadoxine/pyrimethamine) in 1985 (Thaithong et al, 1988;Labbe et al, 2001). Several studies have shown that more than 50% of Thai isolates are resistant to MQ in vitro, and that the prevalence of the D10-like N1042 and D1246 pfmdr1 polymorphisms is greater than 90% (Price et al, 1999;Lopes et al, 2002;Price et al, 2004), consistent with both a fitness advantage in the absence of CQ and previous observations associating MQ resistance with these polymorphisms (Duraisingh et al, 2000b;Reed et al, 2000).…”

“…In some parts of south-east Asia and most of South America, polymorphisms associated with resistance in one or both these genes are near-ubiquitous (Povoa et al, 1998;Zalis et al, 1998;Labbe et al, 2001;Vieira et al, 2001;Contreras et al, 2002;Cortese et al, 2002;Lopes et al, 2002;Vinayak et al, 2003;Casey et al, 2004;Huaman et al, 2004;Vathsala et al, 2004), a situation attributed to relatively higher levels of symptomatic infections and consequently increased levels of drug usage (Warhurst and Duraisingh, 2001;Hastings, 2003). In such circumstances, cessation of CQ use would not automatically lead to an expanded population of parasites carrying the wildtype alleles as the possibility of mixed infections, and thus competition between resistant and sensitive parasites, would be minimal.…”